Plant virus diseases can damage leaves, stems, roots, fruits, seed, or flowers, and are responsible for a considerable percentage of economic loss due to reduced crop yield and quality (Agrios, “Chapter 14: Plant Diseases Caused By Viruses,” in Plant Pathology, 3rd Ed., San Diego, Calif.: Academic Press, p. 655 (1988)).
Control of plant virus diseases took a major step forward when it was shown that the tobacco mosaic virus (“TMV”) coat protein (“CP”) gene that was expressed in transgenic tobacco conferred resistance to TMV (Powell-Abel et al., “Delay of Disease Development in Transgenic Plants that Express the Tobacco Mosaic Virus Coat Protein Gene,” Science 232:738-43 (1986)). The concept of pathogen-derived resistance (“PDR”), which states that pathogen genes that are expressed in transgenic plants will confer resistance to infection by the homologous or related pathogens (Sanford et al., “The Concept of Parasite-Derived Resistance—Deriving Resistance Genes from the Parasite's Own Genome,” J. Theor. Biol., 113:395-405 (1985)) was introduced at about the same time. Since then, numerous reports have confirmed that PDR is a useful strategy for developing transgenic plants that are resistant to many different viruses (Lomonossoff, G. P., “Pathogen-Derived Resistance to Plant Viruses,” Ann. Rev. Phytopathol., 33:323-43 (1995)). However, additional modes of protecting plants against virus disease are needed.
Recessive disease resistance genes are widely deployed in agriculture and are common in nature. However, not much is understood about the identity of naturally occurring recessive disease resistance genes in plants. Plant disease resistance conferred by recessive genetic factors has received limited attention relative to dominant R genes, despite their durability and prevalence in nature. Thus, there is a need to identify, isolated, and clone plant host genes whose gene product is essential for pathogenesis. This knowledge would be useful in that it would enable one to engineer disease resistance the various gene silencing methods available in the art.
Potyviruses comprise approximately 30% of all known plant viruses and as a group are very destructive in agriculture (Ward et al., “Taxonomy of Potyviruses: Current Problems and Some Solutions,” Intervirology 32:269-296 (1991)). The family Potyviridae is characterized by a monopartite single-stranded positive sense RNA genome with a covalently-bound viral-encoded protein (VPg) attached at the 5′ terminus and a 3′ poly-A tract (Riechmann et al., “Highlights and Prospects of Potyvirus Molecular Biology,” J. Gen. Virol. 73:1-16 (1992)). The genome is approximately 10 kb in length and is translated as a polyprotein which is subsequently cleaved into smaller polypeptides by viral-encoded proteases. Based on similarities in genome structure, including conserved order and function among homologous replication proteins, potyviruses have been assigned to the proposed picorna-like superfamily of viruses, which includes many important human and animal pathogens, such as poliovirus and foot-and-mouth disease virus (Goldbach et al., “Genetic Organization, Evolution and Expression of Plant Viral RNA Genomes,” In Fraser, ed., Recoginition and Response in Plant-Virus Interactions, Heidelberg:Springer-Verlag, pp. 147-162 (1990); and Riechmann et al., “Highlights and Prospects of Potyvirus Molecular Biology,” J. Gen. Virol. 73:1-16 (1992)).
Potyvirus infection requires the interaction of host factors with viral proteins and RNA for replication and systemic spread (Carrington et al., “Cell-to-Cell and Long-Distance Transport of Viruses in Plants,” Plant Cell 8:1669-1681 (1996)). Although much is known regarding the functions of the individual potyvirus proteins and RNA structures in viral replication and movement (reviewed in Revers et al., “New Advances in Understanding the Molecular Biology of Plant/Potyvirus Interactions,” Mol. Plant Microbe Interact. 12:367-376 (1999); Riechmann et al., “Highlights and Prospects of Potyvirus Molecular Biology,” J. Gen. Virol. 73:1-16 (1992); and Urcuqui-Inchima et al., “Potyvirus Proteins: A Wealth of Functions,” Virus Res. 74:157-175 (2001)), very little is understood about the identity and functions of host factors that are required for potyviral infection. Towards this end, the identification of naturally occurring host mutations that result in viral resistance and display monogenic recessive inheritance should define an important resource. The “negative model” of plant virus resistance predicts that a recessive resistance gene may represent a deleted or defective host protein that is essential for viral infection but is dispensable for the host (Fraser, “The Genetics of Plant-Virus Interactions: Implications for Plant Breeding,” Euphytica 63:175-185 (1992)). Recessive resistance is especially prevalent for potyviruses, comprising approximately 40% of all known resistance genes (Provvidenti et al., “Sources of Resistance to Viruses in the Potyviridae,” Arch. Virol. 5:189-211 (1992)). Many of these genes, including the Capsicum resistance gene pvr1, have been used successfully for decades in crop breeding programs as effective and stable sources of resistance (Greenleaf, “Pepper Breeding,” In Basset, ed., Breeding Vegetable Crops, Westport, Conn.: AVI Pub., pp. 67-134 (1986)).
The potyviral NIa protein, also known as VPg-Pro, is comprised of an N-terminal VPg and C-terminal protease and participates in several replicative and proteolytic functions during potyvirus infection (Revers et al., “New Advances in Understanding the Molecular Biology of Plant/Potyyirus Interactions,” Mol. Plant Microbe Interact. 12:367-376 (1999)). The central region of VPg has been shown to be crucial in race-specific replication, cell-to-cell and long-distance movement functions in relation to recessive potyvirus resistance genes (Keller et al., “Potyvirus Genome-Linked Protein (VPg) Determines Pea Seed-Borne Mosaic Virus Pathotype-Specific Virulence in Pisum sativum,” Mol. Plant Microbe Interact. 11:124-130 (1998); Masuta et al., “A Single Amino Acid Change in Viral Genome-Associated Protein of Potato Virus Y Correlates with Resistance Breaking in ‘Virgin A Mutant’ Tobacco,” Phytopathology 89:118-123 (1999); Nicolas et al., “Variations in the VPg Protein Allow a Potyvirus to Overcome va Gene Resistance in Tobacco,” Virology 237:452-459 (1997); Rajamaki et al., “Viral Genome-Linked Protein (VPg) Controls Accumulation and Phloem-Loading of a Potyvirus in Inoculated Potato Leaves,” Mol. Plant Microbe Interact. 15:138-149 (2002); and Schaad et al., “VPg of Tobacco Etch Potyvirus is a Host Genotype-Specific Determinant for Long-Distance Movement,” J. Virol. 71:8624-8631 (1997)). The importance of NIa in potyvirus replication and movement has also prompted studies to identify host factors that interact with this protein using in vitro interaction assays. One study showed strong interaction in yeast two-hybrid assays between Tobacco etch virus (TEV) NIa and translation initiation factor eIF4E isolated from tomato and tobacco (Schaad et al., “Strain-Specific Interaction of the Tobacco Etch Virus NIa Protein with the Translation Initiation Factor eIF4E in the Yeast Two-Hybrid System,” Virology 273:300-306 (2000)). Strong interactions have also been observed between Arabidopsis thaliana eIF4E or eIF(iso)4E and Turnip mosaic virus (TuMV) VPg-Pro both in yeast two-hybrid and ELISA-based in vitro binding assays (Wittmann et al., “Interaction of the Viral Protein Genome Linked of Turnip Mosaic Potyvirus with the Translational Eukaryotic Initiation Factor (iso) 4E of Arabidopsis thaliana Using the Yeast Two-Hybrid System,” Virology 234:84-92 (1997)). Furthermore, the interaction of Arabidopsis eIF(iso)4E and TuMV VPg-Pro correlated with viral infectivity (Leonard et al., “Complex Formation Between Potyvirus VPg and Translation Eukaryotic Initiation Factor 4E Correlates with Virus Infectivity,” J. Virol. 74:7730-7737 (2000)).
The present invention is directed to overcoming these deficiencies in the art.